Transcytosis and coenzymatic conversion of [&lt;sup&gt;57&lt;/sup&gt;Co]cobalamin bound to either endogenous transcobalamin II or exogenous intrinsic factor in Caco-2 cells

Pons, Laurent; Guy, Muriel; Lambert, Daniel; Hatier, R; Guéant, Jean‐Louis

doi:10.1159/000016344

Cited by 23 publications

(37 citation statements)

References 32 publications

(40 reference statements)

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“…Freund (INSERM U.381, Strasbourg, France). Cells were routinely grown as described previously [19]. DMEM medium containing 5 % FCS and no methionine (Met free medium) was supplemented with either 30 mg/l L-methionine (Met+), and/or 50 µM Homocystine (Hcy+), each of them with or without Cbl (Cbl+ or Cbl free) at the concentrations indicated in the text.…”

Section: Culture Conditionsmentioning

confidence: 99%

Homocysteine Increases Methionine Synthase mRNA Level in Caco-2 Cells

Ortiou

Alberto²,

Guéant³

et al. 2004

Cell Physiol Biochem

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Background: Methionine synthase (MTR) synthesizes methionine from homocysteine, using cobalamin as a cofactor and 5-methyltetrahydrofolate as a cosubstrate. Aim: To determine the influence of homocystine (Hcy, dimer of homocysteine) in the presence of either cobalamin or methionine on the transcription and the activity of methionine synthase in Caco-2, a human adenocarcinoma cell line. Methods: Methionine synthase activity and quantification of its mRNA by real-time RT-PCR were determined in cells cultivated under four differents conditions: Hcy with cobalamin (Hcy+ Cbl+), Hcy with methionine (Hcy+Met+), methionine with Cbl (Met+ Cbl+) and methionine only (Met+). Results: Activity (nmol/h/mg protein) was maximal in cells cultivated in Hcy+Cbl+ (2.45 ± 0.35), compared to cells cultivated in Hcy+Met+ (0.18 ± 0.01, p<0.001), in Met+ Cbl+ (1.60 ± 0.06, p<0.05), and in Met+ (0.40 ± 0.05, p<0.001), suggesting an adaptation of the cells to requirement in synthesized methionine. The mRNA level of MTR in Hcy+ Cbl+ and Hcy+Met+ (2.82 ±0.49 and 3.33 ± 0.48 AU, respectively ) was about 2.5 / 3.0-fold higher than that in Met+ Cbl+ and in Met+ (1.00 ±0.13 and 1.20 ±0.20 AU, respectively, p<0.001). Conclusion: Methionine synthase expression of Caco-2 cell is under a transcriptional control influenced by Hcy.

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Section: Culture Conditionsmentioning

confidence: 99%

Homocysteine Increases Methionine Synthase mRNA Level in Caco-2 Cells

Ortiou

Alberto²,

Guéant³

et al. 2004

Cell Physiol Biochem

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“…Until now, the consequences of B12 deficiency in the brain have been difficult to evidence because of the experimental limitations of the classical cell models. Indeed, the minute amount of vitamin B12 needed by cells can be provided in vitro by B12 from the FCS, which can bind the ''autocrine'' B12 carrier protein, transcobalamin (TC) (3,4).…”

mentioning

confidence: 99%

“…The expression of oleosin in reticulum remains when oleosin is heterologously expressed in mammalian cells (6). TC is the protein that displays the highest affinity for B12; it is constitutively secreted into plasma to bind the circulating vitamin B12, internalized into cells via receptormediated endocytosis, and subsequently addressed in cytosol and mitochondria (3,4,7). We linked TC to the hydrophobic oleosin to sequester B12 in the cytosolic face of intracellular membranes.…”

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confidence: 99%

Vitamin B12 deficiency reduces proliferation and promotes differentiation of neuroblastoma cells and up-regulates PP2A, proNGF, and TACE

Battaglia-Hsu

Akchiche

Noel

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Vitamin B12 (cobalamin, Cbl) is indispensable for proper brain development and functioning, suggesting that it has neurotrophic effects beside its well-known importance in metabolism. The molecular basis of these effects remains hypothetical, one of the reasons being that no efficient cell model has been made available for investigating the consequences of B12 cellular deficiency in neuronal cells. Here, we designed an approach by stable transfection of NIE115 neuroblastoma cells to impose the anchorage of a chimeric B12-binding protein, transcobalamin-oleosin (TO) to the intracellular membrane. This model produced an intracellular sequestration of B12 evidenced by decreased methyl-Cbl and Sadenosylmethionine and increased homocysteine and methylmalonic acid concentrations. B12 deficiency affected the proliferation of NIE115 cells through an overall increase in catalytic protein phosphatase 2A (PP2A), despite its demethylation. It promoted cellular differentiation by improving initial outgrowth of neurites and, at the molecular level, by augmenting the levels of proNGF and p75 NTR . The up-regulation of PP2A and pro-nerve growth factor (NGF) triggered changes in ERK1/2 and Akt, two signaling pathways that influence the balance between proliferation and neurite outgrowth. Compared with control cells, a 2-fold increase of p75 NTR -regulated intramembraneous proteolysis (RIP) was observed in proliferating TO cells (P < 0.0001) that was associated with an increased expression of two tumor necrosis factor (TNF)-␣ converting enzyme (TACE) secretase enzymes, Adam 10 and Adam 17. In conclusion, our data show that B12 cellular deficiency produces a slower proliferation and a speedier differentiation of neuroblastoma cells through interacting signaling pathways that are related with increased expression of PP2A, proNGF, and TACE.homocysteine ͉ neurotrophin V itamin B12 (B12, also named cobalamin, Cbl) deficiency has long been associated with pernicious anaemia (1) and neurological disorders that range from minor behavior changes to severe neurodegenerative disorders (2). Molecular mechanisms are still lacking to explain how the deficiency can bring about all of the symptoms observed. Indeed, only two enzymes are B12-dependent in mammalian cells: the mitochondrial enzyme L-methylmalonyl-CoA mutase (EC 5.4.99.2) and the cytoplasmic homocysteine methyltransferase, also referred as methionine synthase (EC 2.1.1.13). Inferences are thus based on the two direct consequences of B12 deficiency: the accumulation of methylmalonic acid (MMA) and homocysteine (Hcy). Until now, the consequences of B12 deficiency in the brain have been difficult to evidence because of the experimental limitations of the classical cell models. Indeed, the minute amount of vitamin B12 needed by cells can be provided in vitro by B12 from the FCS, which can bind the ''autocrine'' B12 carrier protein, transcobalamin (TC) (3, 4).To delineate the role of B12 in neurological disorders, we designed a neuronal cell model made deficient in B12 through the stable ...

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“…In the present study, Caco-2/HT29-MTX co-cultured cell model was used, which consists of both absorptive enterocyte-like Caco-2 cells and the mucus-producing goblet cell-like HT29-MTX cells. It is also confirmed that both of them have an IF-dependent fashion (32-35) and IF-independent fashion (29,(36)(37) for the transportation of VB12. Also, this cell model could be affected by some chemical reagent.…”

Section: Disscusionmentioning

confidence: 53%

Efficient Peroral Delivery of Insulin via Vitamin B12 Modified Trimethyl Chitosan Nanoparticles

Guo

Zhu

et al. 2015

J Pharm Pharm Sci

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ABSTRACT-PURPOSE:We investigated the effect of vitamin B12 (VB12) modification on the insulin absorption from trimethyl chitosan(TMC) nanoparticles (NPs) under the influence of mucus. METHODS: TMC and TMC-VB12 were synthesized and insulin loaded TMC/TMC-VB12 nanoparticles were prepared and characterized. Modified and unmodified nanoparticles were studied with Caco-2/HT29-MTX cell model and ligated rat ileum loop. RESULTS: Compared with unmodified NPs, VB12 modified NPs showed significantly higher drug internalization in Caco-2/HT29-MTX cell model. The internalization mechanism via VB12 mediation included caveolae and clathrin-mediated endocytosis pathway. Meanwhile, an increased transportation of drugs was observed for VB12 modified NPs, possibly due to the ligand-receptor interaction via an intrinsic factor-dependent fashion. Although the uptake and transport of VB12 modified NPs could be partially influenced by mucus, they still showed higher drug permeation through Caco-2/HT29-MTX co-cultured cells than unmodified NPs in the presence or absence of mucus. Moreover, in situ study in ligated rat ileum loop demonstrated that VB12 modified nanoparticles could reduce the residual insulin in intestinal lumen (0.59 times) and increase their absorption in epithelial tissue (4.8 times) compared with the unmodified ones. CONCLUSION: VB12 modified trimethyl chitosan nanoparticle is a promising carrier for peroral delivery of insulin.

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Transcytosis and coenzymatic conversion of [<sup>57</sup>Co]cobalamin bound to either endogenous transcobalamin II or exogenous intrinsic factor in Caco-2 cells

Cited by 23 publications

References 32 publications

Homocysteine Increases Methionine Synthase mRNA Level in Caco-2 Cells

Homocysteine Increases Methionine Synthase mRNA Level in Caco-2 Cells

Vitamin B12 deficiency reduces proliferation and promotes differentiation of neuroblastoma cells and up-regulates PP2A, proNGF, and TACE

Efficient Peroral Delivery of Insulin via Vitamin B12 Modified Trimethyl Chitosan Nanoparticles

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